Translocation control for sensing by a nanopore

ABSTRACT

Translocation control for sensing by a nanopore, as well as methods and products related to the same, are provided. Such methods optimize duplex stability to provide high fill rate (of the hybridization sites) but do not prevent rapid dissociation required for high read rates, as well as controlling the translocation of a target molecule for sensing by a nanopore by use of a selective pulsed voltage. Products related to the same include a reporter construct comprising two or more phosphoramidites.

BACKGROUND Statement Regarding Sequence Listing

The Sequence Listing associated with this application is provided intext format in lieu of a paper copy, and is hereby incorporated byreference into the specification. The name of the text file containingthe Sequence Listing is SEQUENCE_LISTING.txt. The text file is 1.25 KB,was created on Apr. 29, 2020, and is being submitted electronically viaEFS-Web.

TECHNICAL FIELD

This invention is generally directed to controlling the translocation ofa target molecule for sensing by a nanopore, as well as methods andproducts relating to the same.

DESCRIPTION OF THE RELATED ART

Measurement of biomolecules is a foundation of modern medicine and isbroadly used in medical research, and more specifically in diagnosticsand therapy, as well in drug development. Nucleic acids encode thenecessary information for living things to function and reproduce, andare essentially a blueprint for life. Determining such blueprints isuseful in pure research as well as in applied sciences. In medicine,sequencing can be used for diagnosis and to develop treatments for avariety of pathologies, including cancer, heart disease, autoimmunedisorders, multiple sclerosis, and obesity. In industry, sequencing canbe used to design improved enzymatic processes or synthetic organisms.In biology, this tool can be used to study the health of ecosystems, forexample, and thus has a broad range of utility. Similarly, measurementof proteins and other biomolecules has provided markers andunderstanding of disease and pathogenic propagation.

An individual's unique DNA sequence provides valuable informationconcerning their susceptibility to certain diseases. It also providespatients with the opportunity to screen for early detection and/or toreceive preventative treatment. Furthermore, given a patient'sindividual blueprint, clinicians will be able to administer personalizedtherapy to maximize drug efficacy and/or to minimize the risk of anadverse drug response. Similarly, determining the blueprint ofpathogenic organisms can lead to new treatments for infectious diseasesand more robust pathogen surveillance. Low cost, whole genome DNAsequencing will provide the foundation for modern medicine. To achievethis goal, sequencing technologies must continue to advance with respectto throughput, accuracy, and read length.

Over the last decade, a multitude of next generation DNA sequencingtechnologies have become commercially available and have dramaticallyreduced the cost of sequencing whole genomes. These include sequencingby synthesis (“SBS”) platforms (Illumina, Inc., 454 Life Sciences, IonTorrent, Pacific Biosciences) and analogous ligation based platforms(Complete Genomics, Life Technologies Corporation). A number of othertechnologies are being developed that utilize a wide variety of sampleprocessing and detection methods. For example, GnuBio, Inc. (Cambridge,Mass.) uses picoliter reaction vessels to control millions of discreetprobe sequencing reactions, whereas Halcyon Molecular (Redwood City,Calif.) was attempting to develop technology for direct DNA measurementusing a transmission electron microscope.

Nanopore based nucleic acid sequencing is a compelling approach that hasbeen widely studied. Kasianowicz et al. (Proc. Natl. Acad. Sci. USA 93:13770-13773, 1996) characterized single-stranded polynucleotides as theywere electrically translocated through an alpha hemolysin nanoporeembedded in a lipid bilayer. It was demonstrated that duringpolynucleotide translocation partial blockage of the nanopore aperturecould be measured as a decrease in ionic current. Polynucleotidesequencing in nanopores, however, is burdened by having to resolvetightly spaced bases (0.34 nm) with small signal differences immersed insignificant background noise. The measurement challenge of single baseresolution in a nanopore is made more demanding due to the rapidtranslocation rates observed for polynucleotides, which are typically onthe order of 1 base per microsecond. Translocation speed can be reducedby adjusting run parameters such as voltage, salt composition, pH,temperature, and viscosity, to name a few. However, such adjustmentshave been unable to reduce translocation speed to a level that allowsfor single base resolution.

Stratos Genomics has developed a method called Sequencing by Expansion(“SBX”) that uses a biochemical process to transcribe the sequence ofDNA onto a measurable polymer called an “Xpandomer” (Kokoris et al.,U.S. Pat. No. 7,939,259, “High Throughput Nucleic Acid Sequencing byExpansion”). The transcribed sequence is encoded along the Xpandomerbackbone in high signal-to-noise reporters that are separated by ˜10 nmand are designed for high-signal-to-noise, well-differentiatedresponses. These differences provide significant performanceenhancements in sequence read efficiency and accuracy of Xpandomersrelative to native DNA. Xpandomers can enable several next generationDNA sequencing detection technologies and are well suited to nanoporesequencing.

Gundlach et al. (Proc. Natl. Acad. Sci. 107(37): 16060-16065, 2010) havedemonstrated a method of sequencing DNA that uses a low noise nanoporederived from Mycobacterium smegmatis (“MspA”) in conjunction with aprocess called duplex interrupted sequencing. In short, a double strandduplex is used to temporarily hold the single stranded portion in theMspA constriction. This process enables better statistical sampling ofthe bases held in the limiting aperture. Under such conditions singlebase identification was demonstrated; however, this approach requiresDNA conversion methods such as those disclosed by Kokoris et al.(supra).

Akeson et al. (WO2006/028508) disclosed methods for characterizingpolynucleotides in a nanopore that utilize an adjacently positionedmolecular motor to control the translocation rate of the polynucleotidethrough or adjacent to the nanopore aperture. At this controlledtranslocation rate (350-2000 Hz (implied measurement rate)), the signalcorresponding to the movement of the target polynucleotide with respectto the nanopore aperture can be more closely correlated to the identityof the bases within and proximal to the aperture constriction. Even withmolecular motor control of polynucleotide translocation rate through ananopore, single base measurement resolution is still limited to thedimension and composition of the aperture constriction. As such, inseparate work, Bayley et al. (alpha hemolysin: Chemistry & Biology9(7):829-838, 2002) and Gundlach et al. (MspA: Proceedings of theNational Academy of Sciences 105(52):20647-20652, 2008) have disclosedmethods for engineering nanopores with enhanced noise and baseresolution characteristics. However, a demonstration of processiveindividual nucleotide sequencing has yet to be published that useseither (or both) a molecular motor for translocation control and anengineered nanopore. Current state of the art suggests that signaldeconvolution of at least triplet base sets would be required in orderto assign single base identity.

Nanopores have proven to be powerful amplifiers, much like their highlyexploited predecessors, Coulter Counters. However, the currentgeneration of organic nanopores (such as Hemolysin and MspA), that havebeen tasked with base recognition of DNA, are transmembrane proteinsthat do not interact with DNA in nature. They do not have naturalfunctions for controlling DNA translocation. As has been discussed, thisis a recognized shortcoming that some have attempted to correct byadding functionality with protein motors adjacent to the nanopores. Forexample, Akeson's group added phi 29 polymerase adjacent to thealpha-hemolysin nanopore so that ss-DNA could be fed into the pore at acontrolled rate (see G. M. Cherf et al. “Automated forward and reverseratcheting of DNA in a nanopore at 5-A precision,” Nat Biotech, vol.advance online publication, February 2012). This approach complicatesthe assay and imposes a separation of the measurement region in thealpha hemolysin from the position control in the polymerase that canintroduce additional noise and sequence dependent variation to themeasurement.

Translocation control by hybridization (TCH) is used herein to refer toa method to pause a nanopore translocation event by using a structurecreated by hybridization which disassociates for translocation toproceed. Akeson et al. (U.S. Pat. No. 6,465,193) first demonstrated thisby pausing DNA translocation with sequential hairpin duplexed regions.Translocation stopped at the duplex because it was larger than theα-hemolysin nanopore aperture. When the duplex released due tostochastic thermal fluctuation, translocation proceeded to the nextduplex. During each pause, the region of DNA located in the nanopore(adjacent to the duplex) could be measured and identified. Akesonmeasured translocations of molecules with sequential duplexed regionsand identified homogeneous regions adjacent to the duplexes. Heestimated the mean of the stochastic pauses to be to 18 μs per base pairfor hairpin duplexes of 2 to 10 base pairs. Akeson further proposed thatsimilar methods of pausing could be accomplished with alternativenon-DNA structures.

Meller et al. (U.S. Pat. No. 7,972,858) used TCH to sequentially pausethe translocation of the DNA with a series of duplexes formed byhybridizing complimentary oligomers to regions along the DNA. Thistechnique utilized an optical technique to measure the each type ofduplex that was sequentially released.

Gundlach et al. also used complementary oligomers for TCH (which hecalled “duplex interrupted”) to measure DNA designed with multipleduplex regions using a low noise nanopore derived from Mycobacteriumsmegmatis (“MspA”) (Proc. Natl. Acad. Sci., 2010). In this case theduplexes were formed by free strand hybridization and paused an adjacent3-base homopolymer portion in the MspA constriction for measurement.Under such conditions three-base homopolymer identification wasdemonstrated. The complementary oligomers used for duplexing were 14bases long, but had poor hybridization fill rates of only 65%. Forsequencing, this approach requires DNA conversion methods such as thosedisclosed by Kokoris et al. or Meller et al. (supra).

In addition, groups have previously used duplexes to hold and releasemolecules form nanopores for a variety of applications, for example: (i)F. Sauer-Budge et. al., “Unzipping Kinetics of Double-Stranded DNA in aNanopore,” Phys. Rev. Lett., vol. 90, no. 23, p. 238101, June 2003; (ii)N. Ashkenasy, J. Sánchez-Quesada, M. R. Ghadiri, and H. Bayley,“Recognizing a Single Base in an Individual DNA Strand: A Step TowardNanopore DNA Sequencing,” Angew Chem Int Ed Engl, vol. 44, no. 9, pp.1401-1404, February 2005; (iii) S. Howorka and H. Bayley, “ProbingDistance and Electrical Potential within a Protein Pore with TetheredDNA,” Biophysical Journal, vol. 83, no. 6, pp. 3202-3210, December 2002;and (iv) W. A. Vercoutere, S. Winters-Hilt, V. S. DeGuzman, D. Deamer,S. E. Ridino, J. T. Rodgers, H. E. Olsen, A. Marziali, and M. Akeson,“Discrimination among individual Watson-Crick base pairs at the terminiof single DNA hairpin molecules,” Nucleic Acids Res, vol. 31, no. 4, pp.1311-8, February 2003.

While significant advances have been made in this field, commerciallyviable implementation of duplex translocation control with, for example,Xpandomers would benefit from improvements that overcome limitationscaused by duplexing, including: (i) compositions for reporters toprovide low noise, ion current blockage signals with amplitudesdistributed for distinct high signal-to-noise measurements in nanopores;(ii) compositions that provide for uniform TCH release rates; (iii)improving poor hybridization fill rate on Xpandomer TCH duplex sites(ideally approaching 100% since each missed duplex can lead to lostsequence information); (iv) improving or mitigating lost dynamic rangedue to ion current blockage of the duplex at the nanopore entrance;and/or (v) methods to better utilize nanopore measurement bandwidth thatis limited by the stochastics of duplex dissociation.

The present invention fulfills these needs and provides further relatedadvantages as discussed below.

BRIEF SUMMARY

In brief, methods are disclosed for controlling the translocation of atarget molecule for sensing by a nanopore, as well as to productsrelating to the same.

In one embodiment, methods are disclosed that optimize duplex stabilityto provide high fill rate (of the hybridization sites) on the Xpandomerbut do not prevent rapid dissociation required for high read rates.

In another embodiment, a duplex is provided that increases the dynamicrange by reducing the ionic current impedance due to the duplex blockageof the nanopore.

In a further embodiment, the complementary regions of the duplexes arereduced to 5 to 8 base pairs by using G-clamp nucleotide analogs toincrease stabilization reducing Xpandomer length for improvedperformance and reduced costs.

In another embodiment, dissociation rate of the duplexes is controlledby a voltage control technique. This technique increases the sequencemeasurement rate for a given bandwidth by enabling regular clocking ofthe dissociation release overcoming limitations of the stochasticdissociation under constant voltage measurements. In addition, thepredictable release time removes temporal uncertainty of a transitionand simplifies signal analysis methods.

In another embodiment, a method for controlling the translocation of atarget molecule for sensing by a nanopore is disclosed, the methodcomprising passing the target molecule through the nanopore subjected toa base voltage and a pulsed voltage, wherein the target moleculecomprises two or more duplex features which provide translocationcontrol by hybridization. In a more specific aspect of this embodiment,the pulsed voltage is sufficient to release the duplex feature engagedwith the nanopore, while leaving the next duplex feature of the targetmolecule to engage with the nanopore unaffected.

In more specific embodiments, the pulsed voltage has a duration of lessthan 100 microseconds, less than 50 microseconds, less than 10microseconds, less than 5 microseconds, or less than 1 microseconds

In more specific embodiments, the pulsed voltage has a voltage ofgreater than 0.2 volts, greater than 0.5 volts, greater than 1 volt, orgreater than 10 volts.

In more specific embodiments, the periodicity of the pulsed voltageranges from 50 Hz to 10 kiloHz.

In more specific embodiments, the target molecule is sensed by thenanopore during the time period between pulses of the pulsed voltage.

In more specific embodiments, a duplex feature of the target molecule isreleased upon each pulse of the pulsed voltage, and in anotherembodiment is released upon multiple pulses of the pulsed voltage.

In another embodiment, a reporter construct is disclosed comprising alow impedance polymer and a high impedance polymer. Exemplary lowimpedance polymers include triethylene glycol (X), hexaethylene glycol(D), ethane (L), hexane (P), dodecane (Z), and abasic (Q), whileexemplary high impedance polymers include deoxyadenosine (A),deoxythymine (T), deoxycytosine (C), and deoxyguanosine (G).

In a further embodiment, a reporter construct is disclosed comprisingtwo or more phosphoramidites selected from the group consisting oftriethylene glycol (X), hexaethylene glycol (D), ethane (L), hexane (P),dodecane (Z), deoxyadenosine (A), deoxythymine (T), deoxycytosine (C),deoxyguanodine (G) and abasic (Q), and wherein the reporting constructcomprises at least one of X, D, L, P, Z or Q.

In another embodiment, at least a reporter may comprise the backbonewith an additional chemical conjugate that can cause deeper ion currentblockage states. This may be a base-conjugation or may result from abranched phosphoramidite.

These and other aspects of the invention will be apparent upon referenceto the following detailed description. To this end, various referencesare set forth herein which describe in more detail certain backgroundinformation, procedures and/or products, and are each herebyincorporated by reference in their entirety.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1 depicts a representative Xprobe having a 4-base probe with basesequence information encoded in 4 reporter constructs labeled R1, R2,R3, and R4 located along the tether representative Xprobe.

FIG. 2 depicts how representative sequence-specific Xprobes are ligatedby template dependent extension to the template target and form anXpandomer.

FIG. 3 depicts a representative cleaved Xpandomer in the process oftranslocating an α-hemolysin nanopore.

FIG. 4 depicts representative structures for reporters along withseveral types of duplexes.

FIG. 5 depicts a representative G-clamp phosphoramadite that allowsduplexes to be synthesized directly on a synthesizer with specificG-clamp incorporations.

FIGS. 6A and 6B depicts two representative current traces of 6-reportertether translocations.

FIGS. 7A through 7D depicts average duplex release times for reporterconstructs plotted as a function of potential applied across thenanopore.

FIG. 8 depicts homopolymer encoding, with the base sequence for twohexamer Xprobes along the top of the figure and the encoded reporterlevels shown beneath them.

FIG. 9 depicts representative duplex designs.

FIG. 10 depicts hairpin dissociation; namely (for left to right), in thetranslocation pause position, dissociation and folding back onto thetrailing backbone, continuing to translocate the nanopore, and exitingthe nanopore and beginning to refold.

FIG. 11 depicts the current response of a translocation through ananopore.

FIG. 12 depicts several alternate designs that increase ion current flowrelative to the simple free duplex.

FIG. 13 is an electrical schematic that depicts delivery of shortvoltage pulses across a nanopore that minimizes the reactive response ofthe Axopatch™ 200B amplifier (Molecular Devices, CA) headstage. Thenanopore is replaced with an equivalent circuit indicated by capacitorand resistor in parallel.

FIG. 14 depicts a representative voltage pulse applied periodicallyacross the nanopore. Representative pulse periods range from 250microseconds to 10 milliseconds, pulse widths range from 0.5microseconds to 10 microseconds, and pulse amplitudes range from 300 mVto 2.0 V.

FIG. 15 depicts a data trace of relative ion current (I/I_(o)) throughan alpha-hemolysin nanopore as a 6 reporter tether is translocated. Theapplied voltage across the nanopore was 100 mV DC (measured from transto cis), superimposed with a 1.0 V pulse every 2 miliseconds (ms).

FIG. 16A (SEQ ID NOS: 1 and 2) and 16B (SEQ NOS: 2 and 3) depictsymbolic layouts of symmetric tethers having a mix of phosphoramidites,where 2 identical arms of each tether are synthesized from the brancherphosphoramidite depicted as “B”.

FIG. 17 depicts a representative current trace of an Xpandomersynthesized from sequential incorporation of 6 X-NTP with tethers thatare schematically represented in FIG. 16B.

FIG. 18 depicts a tether constructed of 4 reporter constructs and aleader. Each reporter construct has an 11 base duplex site (SEQ ID NO:4) that is indicated along with its complementary duplex (SEQ ID NO: 2).The 4 reporters are each composed of a specific arrangement ofphosphoramidites chosen from triethylene glycol (X), hexaethylene glycol(D), ethane (L) and deoxythymine (T).

FIG. 19 depicts the relative ion current data as a function of appliedvoltage acrosxs the nanopore for each of the 4 reporter constructsdescribed in FIG. 18.

FIG. 20 depicts the release times of the duplexes as a function ofapplied DC voltage (with no pulsing) across the nanopore for each of the4 reporter constructs described in FIG. 18.

FIG. 21 depicts the release times of the duplexes as a function of peakpulse voltage applied at 2 ms intervals (superimposed on a constant 60mV DC baseline) applied across the nanopore for each of the 4 reporterconstructs described in FIG. 18.

DETAILED DESCRIPTION

Sequencing by expansion (SBX) encodes base sequence information intoreporters that are positioned on the tethers of specialized SBX probescalled Xprobes (Kokoris et al). FIG. 1 shows a Xprobe with a 4-baseprobe with base sequence information encoded in 4 reporter constructslabeled R1, R2, R3, and R4 located along the tether. FIG. 2 shows howthe sequence-specific Xprobes are ligated by template dependentextension to the DNA template target and form the Xpandomer. Throughsynthesis of the Xpandomer, the DNA sequence is now encoded along thetether in the ordering of the reporter constructs.

The Xprobe depicted in FIG. 1 has a tether designed for translocationcontrol by hybridization (TCH). As depicted, each reporter construct(R1, R2, R3 and R4) has a region of hybridization which can be duplexedto a complementary oligomer (CO) and is positioned adjacent to areporter. Following the reporter is a connector portion that provides aspacer before the next reporter construct begins. Also shown in thisfigure is the cleavable linker located between the probe bases withtether attachment points. After the Xpandomer is synthesized, thislinker is cleaved under specific conditions which allow the tether toelongate.

Another method of Xpandomer synthesis uses an X-NTP (Kokoris et al.)rather than an Xprobe to extend the template-dependent synthesis anduses polymerase rather than ligase as a catalyst. This method has theadvantage of using a small library of 4 X-NTP relative to a much largerXprobe library (4N where the Xprobe is an N-mer).

FIG. 3 shows a cleaved Xpandomer in the process of translocating anα-hemolysin nanopore. This biological nanopore is embedded into a lipidbilayer membrane which separates and electrically isolates tworeservoirs of electrolytes. A typical electrolyte has 1 molar KClbuffered to a pH of 7.0. When a small voltage, typically 100 mV, isapplied across the bilayer, the nanopore constricts the flow of ioncurrent and is the primary resistance in the circuit. Xpandomerreporters are designed to give specific ion current blockage levels andsequence information can be read by measuring the sequence of ioncurrent levels as the sequence of reporters translocate the nanopore.

A wild-type α-hemolysin nanopore can be described by 2 structuralvolumes along its axis, the vestibule which is 2.8 nm across at itsentrance and widens to 4.8 nm before converging to a 1.5 nm “pinch”point that marks the start of the 2nd volume called the stem. The stemis more cylindrical, ranging from 1.5 nm to 2.6 nm but constricts to 2.2nm at its exit (L. Song, M. R. Hobaugh, C. Shustak, S. Cheley, H.Bayley, and J. E. Gouaux, “Structure of staphylococcal alpha-hemolysin,a heptameric transmembrane pore,” Science, vol. 274, no. 5294, pp.1859-1866, December 1996).

The α-hemolysin nanopore is typically oriented so translocation occursby entering the vestibule side and exiting the stem side. As shown inFIG. 3, the nanopore is oriented to capture the Xpandomer from the stemside first. This orientation is advantageous using the TCH methodbecause it causes fewer blockage artifacts that occur when enteringvestibule first. Unless indicated otherwise, stem side first will be theassumed translocation direction.

As the Xpandomer translocates, a reporter enters the stem until itsduplex stops at the stem entrance. The duplex is ˜2.4 nm in diameterwhereas the stem entrance is ˜2.2 nm so the reporter is held in the stemuntil the complimentary strand of the duplex disassociates (releases)whereupon translocation proceeds to the next reporter. The freecomplementary strand is highly disfavored from entering the nanoporebecause the Xpandomer is still translocating and diffuses away from thepore.

Nanopores may be broadly classified into biological and synthetic types,and both types are intended to be within the scope of this invention.While alpha hemolysin (αHL) is perhaps the most studied biologicalnanopore to date, this and other biological nanopores may be utilized inthe context of this invention, such as Mycobacterium smegmatis porin A(MspA). More recently, synthetic nanopores have been introduced usingpolymers, aluminum oxide, silicon dioxide, silicon nitride or other thinsolid-state membranes. A requirement to use TCH on any nanopore is thatdiameter be restricted at at least one point so the duplexed region ofthe reporter construct will be stopped until the duplex releases. Allsuch design options are within the scope of this invention.

Each reporter construct uses its duplex to position its reporter withina zone of the nanopore that has high ion current resistance. In alphahemolysin, this zone is the stem. In this zone, different reporters aresized to block ion flow at different measureable levels. Specificreporters can be efficiently synthesized using phosphoramidite chemistrytypically used for oligonucleotide synthesis. Reporters can be designedby selecting a sequence of specific phosphoramidites from commerciallyavailable libraries. Such libraries include but are not limited topolyethylene glycol with lengths of 1 to 12 or more ethylene glycolunits, aliphatic with lengths of 1 to 12 or more carbon units,deoxyadenosine (A), deoxycytosine (C), deoxyguanodine (G), deoxythymine(T), abasic (Q). Table 1 below lists some representativephosphoramidites.

TABLE 1 Representative Phosphoramidites Phosphoramidite Short NameSymbol Triethylene glycol PEG-3 X Hexaethylene glycol PEG-6 D Ethane C-2L Hexane C-6 P Dodecane C-12 Z Deoxyadenosine dA A Deoxythymine dT TDeoxycytosine dC C Deoxyguanosine dG G Abasic ab Q Spermine Sp S

Reporter design is guided by balancing measurement characteristicsincluding: (i) normalized ion current (I/I_(o)): where I is ion currentand I_(o) is the open channel current; (ii) ion current noise: includesmulti-state responses, blockages, random spiking, and the like; and/or(iii) release time of the duplex.

Reporter ion current blockage and its duplex release time is alsomodulated by measurement conditions such as: (i) voltage; (ii)electrolyte; (iii) temperature; (iv) pressure; and/or (v) pH.

The duplex associated with the reporter also contributes to the ioncurrent blockage.

For a given set of measurement conditions reporters can be designed fora minimum and maximum I/Io levels that define the measurement dynamicrange. Other reporters can be designed with different I/I_(o) levelswithin the dynamic range. As each reporter is paused in the nanopore,the measured I/I_(o) level must remain stationary long enough and havelow enough noise that the reporter type can be uniquely distinguished.

The maximum I/Io level reporter would ideally have a duplex that blocksminimally followed by a reporter with thin nonblocking cross-section. Incontrast, the minimum I/Io level approaches full blockage with a largecross-section backbone (and optionally a deep blocking duplex). Anadditional requirement is that the reporter clears the pore withrelative ease after the duplex dissociates. It is noteworthy that theblockage is due to a composite resistance formed along the narrowerportions of the nanopore channel.

In one embodiment, each member of a reporter set (following the duplex)is formed by an ordered choice of phosphoramidites that can be selectedfrom many commercial libraries. Each constituent phosphoramiditecontributes to the net ion resistance according to its position in thenanopore (located after the duplex stop), its displacement, its charge,its interaction with the nanopore, its chemical and thermal environmentand other factors. The charge on each phosphoramidite is due, in part,to the phosphate ion which has a nominal charge of −1 but is effectivelyreduced by counterion shielding. The force pulling on the duplex is dueto these effective charges along the reporter which are acted upon bythe local electric fields. Since each reporter can have a differentcharge distribution, it can exert a different force on the duplex for agiven applied voltage. The force transmitted along the reporter backbonealso serves to stretch the reporter out to give a repeatable blockingresponse.

In this example, reporters were designed by choosing phosphoramiditebuilding blocks from deoxyadenosine (dA) and hexaethylene glycol (PEG-6)(ChemGenes, MA)). Four reporters were designed by adding 0, 2, 4, and 6dA in series with the PEG-6 backbone (referred to as OA, 2A, 4A and 6Arespectively). The poly-dA portion is after the duplex so the reporteris positioned in the alpha-hemolysin stem when the duplex stops at thestem entrance. FIG. 4 shows the structure of the 4 reporters along withseveral types of duplexes. For the A4 and A6 reporter the poly-dAportions are AADAA and AAADAAA respectively where a PEG-6 (indicated asD) is placed to tune the ion blocking level response.

The reporter with the maximum I/I_(o) response has 6 PEG-6phosphoramidites in series. Each PEG-6 had a contour length of 1.95 nmincluding its phosphodiester linkage, so the reporter had a totalcontour of 11.7 nm, longer than the alpha-hemolysin pore channel of 10nm. The additional length means that the previous reporter's duplex sitecan translocate beyond the vestibule and minimize any furtherinteraction with the nanopore. In a similar manner the reporters withpoly-dA portions have an additional poly-PEG-6 segment to providevestibule clearance.

Dissociation (or release) of the duplex is a thermal stochastic processthat depends upon multiple factors, including: (i) constituent duplexpairing strengths and order; (ii) applied voltage; (iii) differentialpressure; (iv) electrolyte; (v) temperature; and/or (vi) pH.

A viable sequencing method must have minimal information loss and forsome applications require <10⁻⁴ error rates. Individual reads, though,may have high raw error rates (e.g., ion torrent, MiSeq are ˜1 in 300).By making many redundant measurements of a sequence region, theconsensus of many individual reads can achieve the desired error rate.All errors are not equivalent due to the additional burden they have oninformation processing or discarded measurements. Insertions anddeletions that cannot be localized can seriously degrade the dataquality.

Since a missing duplex is a deletion error, efficient TCH sequencingrequires conditions where duplex fill approaches 100%. Duplex fill isdefined as the ratio of the number of duplexed reporter constructs tothe total number of reporter constructs along the sampled Xpandomers.Short DNA oligomer duplexes of 7 to 8 bases (40 to 50% GC) haveduplexing rates of kon˜1e7 M⁻¹ s⁻¹ at room temperature for 2M saltconditions (S. Howorka, et al. Proceedings of the National Academy ofSciences of the United States of America, vol. 98, no. 23, pp.12996-13001, November 2001) and dissolution rates of koff˜1 s⁻¹. Thusfor a duplex site to be duplexed 99% of the time requires that thekon*M/koff=99 where M is the duplex molarity. This yields a molarityrequirement of M=99/1e7˜10 μM.

Nucleic acid analogs with higher base pairing strengths can reduce theduplex length and increase stability to achieve high duplex fill. Someanalogs that have demonstrated higher melt temperatures than thecanonical DNA base pairs include 2′Methoxy RNA, 2′Fluoro RNA, PNA, LNAand RNA.

A variety of nucleotide modifications located either at the ends orinternal to the oligomer can also be used to modify stability. Forexample a 3′ amino end group helps to stabilize the duplex complementthat approaches a nanopore from the 5′ side.

G-clamps (Isis Pharmaceuticals) are Cytosine analogs that willselectively base-pair to Guanine and will raise thermal melttemperatures significantly. FIG. 5 shows a G-clamp phosphoramadite thatallows duplexes to be synthesized directly on a synthesizer withspecific G-clamp incorporations.

Replacing 1 or 2 cytosine bases with these G-clamps, in an otherwisecanonical base duplex provides substantial increase in stability andfree solution fill rates. FIG. 4 shows the composition of 4 differenthexamer duplexes labeled D1, D2, D3, and D4 that are incorporated intoreporter constructs. The constructs also incorporate 4 reporters A0, A2,A4 and A6. Six reporter constructs were synthesized into longer reportertethers using phosphoramidite chemistry. The 4 reporters block ions in ahemolysin nanopore at 4 different levels. Increasing the number ofadenosines in the nanopore stem increases the charge density, decreasesthe volume through which ions can diffuse, and results in increased ioncurrent resistance. With nanopore buffer of 2 molar NH₄Cl/10 mM HEPES,pH 7.4, and temperature of 20 degrees C., data was logged. FIGS. 6A and6B show two example current traces of 6-reporter tether translocationscaptured at 140 mV. The trace current is shown normalized to the openchannel current which is 182 pA. The order of the six reporters on thetether is A0, A2, A4, A0, A3, A2. As the tether translocates, a duplexstops each reporter in the stem of the nanopore and gives acharacteristic blockage level as shown on the trace. As indicated in thetraces, the relative ion blockages of the four reporters A0, A2, A4, andA6 are 0.270, 0.197, 0.163 and 0.106, respectively. The duplexdissociation times for each reporter type have an exponentialdistribution.

The average duplex release time for each reporter construct is plottedas a function of potential applied across the nanopore in the 4 plots ofFIGS. 7A through 7D. The duplex releases under shear force as it resiststranslocation of the backbone molecule through the nanopore under someapplied voltage. The kinetics of this release has been modeled by theEvans-Kramer escape kinetics (E. Evans, Annual Review of Biophysics andBimolecular Structure, vol. 30, no. 1, pp. 105-128, 2001.) whichpredicts that the axial shear force causes an exponential increase inthe release rate. The force is generated by the electric field actingupon the effective charges of the reporter inside the nanopore andprimarily located in the nanopore stem. Thus by increasing either thepotential or the number of effective charges decreases the averagerelease time of the duplex. Each reporter was synthesized in a constructwith duplex D3 and release times are shown on the plots. The releasetimes of this duplex for A, A2, A4, and A6 at 140 mV are 340, 97, 20 and7 ms showing the effect of increasing charge in the nanopore stem causedby increasing the number of phosphate ions (one for each adenosinebase). The plots show relative stability of the duplexes. Duplexes D1and D3 have 2 and 1 G-clamps respectively but are otherwise identicaland comparing release times shows D1 to be more stable D3 with releasetimes typically 10 times longer (see plots for reporters A4 and A6).Duplexes D3 and D4 each have 1 G-clamp but their G/C contents are 83%and 50% respectively. This also increases stability and release timesare typically 10 times longer (see FIGS. 7A and 7B for plots ofreporters A0 and A2, respectively).

Extrapolation of the duplex release time curves to 0 mV provides anestimate of koff from the inverse of the intercept. These koff valuesranged from 0.3 s⁻¹ to <0.001 s⁻¹, all slower and therefore more stablethan the 7 or 8 canonical base oligomers described by Howorka that hadkoff˜1 s⁻¹.

If duplex fill is not 100%, other coding techniques can be used toprovide redundant measurement or error checking. Xpandomer reportertethers can encode reporters to provide additional functionality thatreduce errors.

Like other sequencing methods, nanopores have difficulty resolvinghomopolymers. There is no inherent signal that will separate the ioncurrent level responses of two consecutive identical bases (orreporters) as they slip through the nanopore.

Xpandomers can encode sequence information along its tether that avoidsthe homompolymer problem. The dynamic range of the nanopore TCH blockagecurrent can resolve 4 to 8 levels. In one coding embodiment, a blockagelevel (and the associated reporter) is assigned for each base type and a5^(th) level is used between identical adjacent bases and also at theend of each Xprobe tether. This forces a transition to occur between any2 states but can increase the number of reporters per Xprobe to avariable number. For example in a tetramer Xprobe, the number ofreporters can vary from 5 to 8 reporters depending upon the number ofhomopolymers.

Another embodiment is to assign a fifth ion blockage level tohomopolymers (Hp). When there is a homopolymer sequence in the probe, itis encoded by assigning the correct base level reporter for the firstbase and then assigning the Hp reporter to the next. Any subsequentconsecutive bases of the same type continue to switch between these twolevels. To decode this, the base type for each Hp level measurement isdetermined by the previous base. FIG. 8 shows the base sequence for 2hexamer Xprobes along the top. The encoded reporter levels are shownbeneath them. An additional 6th ion blockage state called the clock(clk) level is added at the end of each Xprobe. This performs twofunctions. It provides an additional error check to inform the decoderthat six levels should be read between clks and it resets thehomopolymer coding between Xprobes. In this case five guanasine bases(G) fall across the two Xprobes. The clk level forces a transitionbetween the third G at the end of the 1st Xprobe, and the first G of thesecond Xprobe.

In the case of the X-NTP synthesized Xpandomer, the tether only encodesfor a single base. In one encoding scheme, a clock reporter is added tofollow every base-type reporter to provide homopolymer discrimination.

An alternative method to TCH for slowing/stopping the reporter in thenanopore is to use steric hindrance of the reporter itself as itpropagates through the nanopore. Mitchell conjugated an internal base ofa 27-base DNA oligomer with different chemical tags and showed how theseslowed the translocation times through a nanopore (Mitchell and Howorka,“Chemical Tags Facilitate the Sensing of Individual DNA Strands withNanopores 13,” Angewandte Chemie International Edition, vol. 47, no. 30,pp. 5565-5568, 2008.) Mitchell claimed the TCH was constrained to ananopore size range whereas the steric hindrance method could be adaptedto larger sized pores. The steric hindrance reporters were shown to havedeep ion current blockage states and may be used in conjunction with TCHto achieve deep states.

It is clear that other coding methods can be applied to reduce errorsincluding cyclic redundancy codes that allow for 1 or more base callerrors and still recover the correct information (Peterson, W. W. andBrown, D. T. (January 1961). “Cyclic Codes for Error Detection”.Proceedings of the IRE 49(1): 228-235).

The literature discusses two duplex designs that have been used for aTCH molecule; namely, referred to herein as the in-line hairpin andsimple free duplex. These and other designs are shown in FIG. 9 anddiscussed individually below (the following numbers correspond to thedesignation numbers shown in FIG. 9).

(1) The “in-line hairpin” structure is so defined because the THCmolecule's backbone (e.g., Xpandomer) passes through it. This structure,however, results in undesired blockages in an alpha-hemolysin duringstem-first translocations, which is attributed to the hairpin causingthe backbone to refold onto itself within the vestibule and not beingable to exit.

(2) The “foldback hairpin” structure is a novel design that reducesentanglement and vestibule blockage issues by maintaining a linearorientation of the backbone. This is synthesized by branching thebackbone at the loop midpoint using a pendant base conjugation to extendthe backbone. In this case when the hairpin dissociates, it will unfoldand align with the trailing backbone polymer that is sufficiently thinto allow continued translocation. FIG. 10 shows this progression in 4steps, from hairpin i) in the translocation pause position, ii)dissociation, iii) folding back onto the trailing backbone andcontinuing to translocate the nanopore, iv) finally exiting the nanoporeand beginning to refold. The nanopore response to the foldback positionin step iii) can be a deep current state that can be used advantageouslyto discriminate sequentially identical reporter states (withsequentially identical current blockage levels). Choices of molecularbuilding materials and other conjugation positions along the hairpin canbe used to adjust this response.

(3) The “pendant hairpin” is a variant of the foldback hairpin that itsimplifies synthesis. In this case the primary backbone is synthesizedand the monomer consisting of a sequence complimentary to the duplexsite is linked with a conjugation chemistry such as “Click” chemistry.The pendant monomer has sufficient steric mobility that it can hybridizewith the duplex site. The structural difference in this design from thefoldback hairpin is that the loop is replaced with a connector chemistryof suitable length.

(4) The “chimera hairpin” is a hairpin variant that can be assembledwith unbranched synthesis. The backbone for this design passes throughone strand of the duplex in the 3′ to 5′ direction continues through aconnector chemistry such as PEG doubling back to the 5′ end of the otherduplex strand and continuing up through this strand and on to the nextreporter section. This synthesis requires that the direction ofsynthesis flip between 3′->5′ to 5′->3′ and back again as the moleculeis built.

(5) The “simple free duplex” is effective but may result in poor filldefined by the average fraction of duplex sites along the measuredbackbone that are unduplexed during measurement. To maintain high fillrate during translocation, the duplex requires a high kon/low koff and asufficient background concentration of the complementary oligomer.

(6) The “circularized free duplex” also has a detached duplexingoligomer similar to the simple free duplex except that the duplexingoligomer is circularized. It can have 2 or more hybridization sites onthe same molecule. Also the circularization impedes the detachedoligomer from translocating the nanopore (hemolysin). This can restrictthe molecule to 1 reservoir side and eliminates associated backgroundtranslocation noise. Another feature is that its interaction with thepore forces the leading base pair in the duplex to open because thebackbone continues to translocate whereas the circularized complementmust follow the preceding bases back around. This can facilitate theduplex release at lower forces and prevents the lead base pair of theduplex from jamming into the pore.

(7) The “free hairpin duplex” is also similar to the simple free duplexexcept the duplexing oligomer has a hairpin extending from the leadingbase. This hairpin facilitates a lower force release of the duplexsimilar to the circularized free duplex. To prevent translocation itcould also have an additional hairpin on its trailing end.

(8) The “anchored duplex” has a detached duplex oligomer that istethered to an anchor. This anchor is a large cross-section molecule,molecular complex or nanoparticle. This requires a nanopore geometrythat accepts the duplex to proceed but stops further translocation whenthe anchor cannot proceed. FIG. 9 shows an anchored duplex stoppedduring translocation of an alpha-hemolysin pore. In this case theanchor, indicated by the circular object, is stopped at the entrance tothe vestibule which prevents further translocation due to the duplexstretched out in the vestibule. This design approach is also appropriatefor other types of nanopores with large barrels that can acceptdouble-stranded nucleic acid, but do not allow the anchor to pass. Thisleads to other types of coding which can use the length or compositionof the duplexing oligomer (that is tethered to the anchor) in order toproduce the measured signal due to its presence in the nanopore barrel.

The following examples are provided for purpose of illustration, and notfor limitation.

EXAMPLES Example 1 Foldback Hairpin

An experiment was performed that used the foldback hairpin duplex toperform TCH. A molecule was synthesized with 4 foldback hairpins toperform TCH for reporter segments ordered as: poly-A, poly-C6, poly-Aand a poly-PEG6. In the case of the internal poly-A reporter, aconnector of poly-PEG6 was added before the next hairpin. This ensuredthat in each case, when the first three hairpins opened up and began totranslocate, they unfolded back along a “thin” backbone polymer (eitherpoly-C6, poly-PEG6). This unfolded condition was still thin enough totranslocate the nanopore constriction.

The experiment used 2M KCl with 10 mM HEPES at Ph 7.4 in two reservoirsseparated by a PC lipid bilayer membrane that was supported on a 25micron Teflon aperture (see Nahid Jetha et al. “Forming an α-HemolysinNanopore for Single-Molecule Analysis,” Micro and Nano Technologies inBioanalysis, vol. 544, R. S. Foote and J. W. Lee, Eds. Totowa, N.J.:Humana Press, 2009, pp. 113-127). A single WT alpha-hemolysin nanoporewas embedded in the membrane. Open channel ion current was measured tobe 247 pA under a voltage of 120 mV applied between Ag/AgCl electrodespositioned in each reservoir. The electrodes were connected with shortleads to a CV 203BU head stage that connected to a Axopatch™ 200Bamplifier (Molecular Devices, CA). Data was filtered with theamplifier's 10 kHz filter and digitized for data capture at 100ksamples/s. The 4 inline hairpin molecule was introduced to the Cisreservoir to translocate the nanopore vestibule-first.

FIG. 11 show the current response of a translocation. The trace plotscurrent in the vertical axis (normalized to open channel current) vstime expressed in milliseconds along the horizontal. The schematic ofthe molecule shows the response as each hairpin pauses at theconstriction point in the vestibule. The variable ion current blockagelevel depends upon the reporter segment that is paused in the stem. The4 reporters poly-A, poly-C-6, poly-A and a poly-PEG-6 correspond to 4normalized current levels of 0.15, 0.28, 0.15 and 0.24. Note that attransitions between any 2 levels there is a deeper spike in the currentthat drops below 0.1. This corresponds to the blockage when the hairpinunfolds and must translocate its trailing edge overlapped with “thin”PEG6. This deep spike transition can be used to differentiate sequentialstates especially when the consecutive states are the same level. Notethat in the terminal hairpin case there is no overlap and no deepercurrent spike occurs.

Example 2 Low Blocking Duplex

Dynamic range of the reporters is limited by the ion current blockingdue to the duplex. Some duplex designs can reduce such blockage andincrease dynamic range. FIG. 12 shows several alternate designs thatincrease ion current flow relative to the simple free duplex. In oneembodiment, a porous structure or layer is placed in front of thenanopore which stops the duplex (and translocating molecule) but doesnot seriously impede the ion current flow. In another embodiment theduplex is a circularized free oligomer which reduces the distance thatthe end of the duplex can enter the nanopore and thus reduces ioncurrent blockage. In the last embodiment the duplex is a single oligomerwith 2 hairpins that fold back tightly to each other but have anadditional overhang that forms the complementary portion that hybridizesto the translocating molecule.

Example 3 Voltage Control

The distribution of release times at a fixed voltage is generallyexponential (attributed to a single simple release mechanism). Thislimits detection efficiency because data must be captured at a ratesufficient to measure the ion blockage level before the fastest release,but then must wait or capture excess data for all the other slowerduplex releases. According to this distribution, if a sampling rate ischosen so 99.9% of the duplex releases are slow enough to measure atleast one complete sample, then for average measurement time is 1000samples long.

One embodiment used to improve this efficiency is to use redundantreporters. If a sampling rate is chosen so 99.9% of redundant reporterpairs have at least 1 of their 2 duplexes release slow enough to measureat least one complete sample, then for average measurement time perreporter is reduced to 32 samples long (64 samples for the redundantreporter pair). This technique can be extended to 3 or more redundantreporters to get additional improvements but at the expense of furtherextending the Xpandomer length.

In another method a force is selectively applied to the duplex at thenanopore for a short enough period that the duplex is stripped off butthe next duplex that engages with nanopore is unaffected (i.e., does nothave the force applied until the next selective application) or is nolonger affected. The selective force can be delivered in different ways.

In one embodiment the selective force is applied using short,high-voltage pulses. During measurement the voltage is sufficiently lowthat the duplex remains stable but is high enough that thesignal-to-noise is sufficient for measurement and identification of thereporter type. The slopes of the curves in FIG. 7 range from 0.19 dBt/mVfor the A0 reporters to 0.37 dBt/mV for the A6 reporters (where dBt arethe units for Rt=10 log(t1/t2) and t1 and t2 are release time values).Extrapolating linearly to voltages >300 mV, average duplex release timesare in the microsecond regime.

Xpandomers can be designed so that a high voltage microsecond pulsesufficient to release a duplex will be complete before the Xpandomertranslocates and stops at the next duplex. Increasing distance, reducingcharge and providing frictional elements to slow the translocationbetween reporter duplexes can be adjusted to achieve this objective. Byusing this technique, after the pulse is finished, an incoming reporterconstruct duplex engages the nanopore under low voltage and the duplexis stable for measurement. After a measurement period another highvoltage pulse is applied to release the duplex and translocate to thenext duplex. Ideally the duplex release occurs at each pulse, but incases where the pulse fails to release the duplex, the reporterundergoes one or more measurement/pulse cycles until release occurs.

In this example, tethers had 6 duplexes along the backbone with orderedstates A0, A4, A5, A6, A2 and A5. The experimental setup shown in FIG.13 is as described by Jetha except that the ground return to theheadstage was modified to pass through an inductor coil of a 603-2current sense transformer (West Coast Magnetics, CA). The other coil inthe transformer was connected to an 33120A function generator (HewlettPackard, CA) and driven with a fast rising 10 V pulse that immediatelydecayed in several microseconds, once every 2 ms. The output pulse thatappeared across the transformer output was a similar shaped pulse of 1 Vamplitude. Its decay was adjusted at the input to provide a FWHM of 3.4μs. The Axopatch™ 200B amplifier (Molecular Devices, CA) was run with100 mV DC applied to the headstage output, so the voltage applied acrossthe nanopore was 100 mV DC plus a 1 V pulse applied once every 2 ms.

The oscilloscope trace, FIG. 14 shows the voltage pulse (1.0 volt)applied to the nanopore.

The trace shown in FIG. 15 indicates the 6 duplexed states along withunduplexed entry and exit levels. Each duplex release coincided with oneof the pulses that occurred at 2 ms intervals. The middle 4 states wereeach 2 ms long on the first and last states required an additional pulseto release their duplexes.

Periodic release of the duplexes increases the efficiency of measurementbecause the shortest release can be timed to be the minimum periodrequired to acquire a reliable measurement. There is an additionalefficiency in the design of the detector. Now measurements can besynchronized to the pulse frequency to as low as 1 per period, thoughmore may be taken for quality purposes. A related efficiency is gainedin analyzing the data afterwards because states will transition during apulse, so state calling is simplified.

In another embodiment of controlled duplex release, the TCH complementhas a small diameter gold nanocrystal (e.g., 1.4 nm) tethered covalentlyto its end so it lies distal to the nanopore when it is stopped duringtranslocation of the reporter tether. After the reporter ion currentblockage is measured, a short pulse of high frequency (e.g., 1 GHz)magnetic field is applied which couples to the gold particle anddestablilizes the duplex that is already the voltage applied stress. Thetechnique of opening duplexes was demonstrated by K. Hamad-Schifferli,et al. (Nature, vol. 415, no. 6868, pp. 152-155, January 2002), but hasnot been used in conjunction with nanopores.

Each of these methods with periodic clocks are well suited toarray-based nanopores because they can be applied across all poressimultaneously. In the case of the voltage pulse technique the pulse canbe applied across a common electrode.

Alternatively, if feedback were applied to each nanopore, forces couldbe applied continuously or pulse-wise until a release was detected andthe force was removed to measure the next reporter.

Example 4 Flossing

The concept of flossing, reported in 2004 (Sánchez-Quesada, A.Saghatelian, S. Cheley, H. Bayley, and M. R. Ghadiri, “Single DNARotaxanes of a Transmembrane Pore Protein,” Angewandte ChemieInternational Edition, vol. 43, no. 23, pp. 3063-3067, 2004), provides amethod to efficiently read a single Xpandomer multiple times andestablish a low error rate/read. This can be implemented multiple ways.

In one embodiment the Xpandomer sample is introduced on the cis side ofthe nanopore with no duplexes attached to the reporter construct sitesand the free solution duplex complements are introduced on the transside. The Xpandomer is terminally modified with a highly stable hairpin(or duplex) and has another high stability duplex site (initiallyunduplexed) on the leader. Under a negative potential, the Xpandomerleader is captured and the Xpandomer rapidly translocates the nanoporeuntil it is stopped at the terminal hairpin. The voltage is quicklylowered before the hairpin dissociates, and prevents the Xpandomer fromexiting. With the Xpandomer now in the trans-side, the duplexcomplements can now associate with the reporter construct sites alongthe Xpandomer. Also, the specific high stability leader duplex is nowformed which prevents the Xpandomer from exiting from the leader side.The nanopore potential is now inverted and the Xpandomer is measured asit reverses out of the nanopore, stopping to release each reporterduplex, until the leader duplex is reached which will not release. Next,the potential is again inverted and the Xpandomer rethreads the nanoporefor a second cycle of measurement. This can be repeated many times toestablish a high fidelity consensus measurement.

To exit the nanopore after measurement, the Xpandomer is translocated tothe terminal hairpin and a high voltage pulse is applied to release thehairpin. The Xpandomer exits to the trans-side and another Xpandomer iscaptured from the cis-side. One choice for the Xpandomer leader duplexis the fold-back hairpin type shown in FIG. 9 (alternative 3). In thistype of hairpin, the leader will thread through the nanopore after thehairpin is released but when reforming, it prevents reversetranslocation. In another embodiment, symmetric reporter constructs aredesigned to be read in either direction so that a reporter is positionedin the nanopore stem when the duplex stops in either direction. In thiscase the free solution duplexes are in both reservoirs and Xpandomer ismeasured in both translocation directions. In-line hairpins arewell-suited for reporter construct duplexes when doing bidirectionalmeasurement because of their inherent symmetry.

Creating arrays of biological nanopores is optimal when only onenanopore is inserted into the membrane that isolates each trans and ciswell (often either the cis well is common to all nanopores). Inaddition, the ion current blockage often depends upon the direction anXpandomer translocates through the nanopore. For example, the blockagecurrents can be different if a reporter enters from vestibule side orfrom the stem side of a hemolysin nanopore. This means that controllingthe orientation of insertion of the nanopore in the membrane can affectperformance. If flossing is used, the nanopore orientation is lessimportant because the Xpandomer can be measured in either direction andmore specifically in the preferred direction.

Example 5 Symmetric Tether

An example of a Symmetric tether is shown in FIG. 16A The figureindicates a tether that is synthesized using phosphoramidite chemistrystarting from a brancher shown as “B”. Assembly proceeds along 2branches with symmetric additions of the phosphoramidite monomers. Thetwo branches are identical and are indicated in the figure with the 5′to 3′ branch in blue (left of “B”) and the 3′ to 5′ in black (right of“B”). Complements to the branch duplex sites are also shown. The tethersymmetry means it looks and measures the same when it is flippedend-to-end. This attribute means that if flossing is used formeasurement an Xpandomer with symmetric tethers can be measured whentranslocated in either direction. Generally, the final synthesis step ofan X-NTP is to conjugate each end of a tether specifically to each oftwo conjugation points on a modified NTP construct. Synthesis with thesymmetric tether eliminates the need for the conjugations to be specificwhich simplifies the process.

FIG. 16B shows another symmetric tether. Compared to the tether in FIG.16A, this tether has a different reporter code and also has a third arm,LTTT that is synthesized before the brancher phosphoramidite isattached. This third arm contributes to a deep blockage state when itfolds onto the backbone during translocation of a hemolysin nanopore andis stalled in the nanopore barrel by the second duplex along the tether.The deep state can be used advantageously as a clock signal todiscriminate between homopolymer bases. This tether construct wasincorporated into a cytosine X-NTP. DPo4 polymerase was used to extend aprimer to complement a 10-base guanidine homopolymer template. ThisXpandomer extension product was cleaved in an acid solution and itsduplex sites were hybridized to the 10-base complements shown in FIGS.16A and 16B. The Xpandomer was then measured in a hemolysin nanoporeunder 85 mV applied potential. Both reservoirs had 10 mM HEPES and pH7.4, with 2.0 molar NH₄Cl on the trans reservoir and 1.0 molar NH₄Cl onthe cis reservoir. FIG. 17 shows the translocation of one of theseXpandomers with 6 X-NTP incorporations. As each tether translocates thenanopore the reporter state of I/I_(o)=0.30 is measured followed by adeep state of I/I_(o)=0.04. The high state is due to the low ionblocking cross-section of the string of 7 PEG-3 phosphoramidites(XXXXXXX in FIG. 16B) preceding the first duplex into the nanopore. Thelow state is due to high ion blocking when the third arm folds back onthe backbone (LTTT folds at the brancher B onto the TTTT in FIG. 16B) asit precedes the second duplex into the nanopore. Here the deep state isa clock that distinguishes 6 consecutive identical reporter states.

Example 6 X-NTP Tether

X-NTPs are designed with a tether comprising a single reporter selectedfrom 4 possible states, a pendant hairpin for TCH and followed by asection of PEG. Xpandomers are formed by template dependent polymeraseextension of these X-NTPs. A hemolysin nanopore measurement system isprepared with 50 microliters of 2 M KCl in a 7.0 pH aqueous buffer inboth cis and trans reservoirs. 500 fmoles of Xpandomer sample with thesame buffer is added to the cis well. A voltage is applied between cisand trans reservoirs that is 60 mV DC with 1V pulses at 2 ms periods.Each pulse rises rapidly to the 1 volt maximum and decays to the DClevel with a 2 microsecond exponential fall time. Xpandomer translocatesthe nanopore rapidly but stops at each pendant hairpin along its length.At each stoppage, the ion current blockage level of the associatedreporter is measured under the 60 DC applied voltage, identifying thebase of the incorporated X-NTP. When the periodic pulse is applied, thetranslocation force increases and opens pendant hairpin, folding backthe pendant group onto a trailing PEG section which are pulled throughthe nanopore together until another pendant hairpin stops thetranslocation. The pulse is short enough that when the next pendanthairpin engages with the nanopore, the voltage is back to 60 mV DC.

Example 7 Alternative TCH Reporters

A variety of other TCH reporter structures have been synthesized usingthe phosphoramidite monomer set described in Table 1 above. The exampledescribed here used a construct synthesized on a MerMade 12(Bioautomation, TX) with 126 phosphoramidite monomers. The sequence isshown in FIG. 18 and contains a leader portion T8 Z4 L25 followed by 4reporter constructs. The leader facilitates threading of the constructthrough the nanopore. The duplex site on each reporter construct is an11 base sequence that will hybridize to its 11 base complement toprovide TCH for the reporters. The experiment used buffers of 1M NH4Clwith 10 mM HEPES at Ph 7.4 in the cis reservoir and 2M NH4Cl with 10 mMHEPES at Ph 7.4 in the trans reservoir. The reservoirs were separated bya lipid bilayer membrane that was supported on a 25 micron Teflonaperture using a fixture described by Jetha.

A single WT alpha-hemolysin nanopore was embedded in the membrane. Openchannel ion current was measured to be 247 pA under a voltage of 120 mVapplied between Ag/AgCl electrodes positioned in each reservoir. Theelectrodes were connected with short leads to a CV 203BU head stage thatconnected to a Axopatch™ 200B amplifier (Molecular Devices, CA). Datawas filtered with the amplifier's 10 kHz filter and digitized for datacapture at 100 ksamples/s. Full translocation events were indicated inthe data by current blockages with the 4 state levels as expected. Theseevents were analyzed to determine the average relative ion currentblockage (I/I_(o)) and average lifetime for each of the four states.This data is presented as a function of applied voltage across thenanopore in FIG. 19 and FIG. 20.

As shown in FIG. 21, additional data was captured in the sameexperimental conditions using an applied voltage of 60 mV and an appliedvoltage pulse. Pulses were applied every 2 ms by programming theAxopatch™ 200B (Molecular Devices, CA) directly with a programmed pulse(without the use of the transformer shown in FIG. 14). The programmedpulse was triangular in shape with a rise and fall ramp rate of 20mV/microsecond and the pulse length is determined by the peak pulsevoltage. The time constant for the duplex release of these reporters is˜10 ms with the 240 mV peak pulse and 60 my DC. An average release timeof 10 ms means that on average it required five of the pulses to releasethe duplex.

FIGS. 4 and 18 depict sets of reporters that are designed to produce ioncurrent signals in alpha hemolysin nanopores that are easilydifferentiated from each other and that maximize the use of the dynamicrange. Dynamic range is maximized by selecting a backbone of lowimpedance molecules (polymers), typically those with small physicalcross-sections and low linear mass densities. When a reporter iscomposed of only these molecules, the I/I_(o) is the highest. I/I_(o)levels for poly-PEG6 (FIG. 6A-B), poly-C-6 (FIG. 11) and poly-PEG-3(FIG. 19) were 0.27, 0.28, and 0.29-0.33, respectively. In each casethese values also depend upon other experimental conditions,electrolytes, TCH duplex, and the like, but these reporters establishthe highest I/I_(o) in the dynamic range. This low impedance group ofmolecules includes polyethylene glycol, poly-abasic, and poly aliphaticpolymers. To produce lower I/I_(o) levels that range down to <0.1,molecules are chosen from the group of high impedance molecules (thosewith large physical cross-sections and high linear mass density),including A,C,G and T. Positioning additional high impedance moleculesin the backbone of low impedance molecules increasingly lowers theselevels.

It should also be noted that while these THC reporter constructs andmethods have been described in the context of Xpandomers, they have moregeneralized use in labelling of analytes in many other applications,including (for example, biological or nanoparticle labelling assays.

Exemplary embodiments include the following:

Embodiment 1. A method for controlling the translocation of a targetmolecule for sensing by a nanopore, comprising passing the targetmolecule through the nanopore subjected to a base line voltage and apulsed voltage, wherein the target molecule comprises two or more duplexfeatures which provide translocation control by hybridization.

Embodiment 2. The method of embodiment 1, wherein the two or more duplexfeatures which provide translocation control by hybridization areselected from the group consisting of an in-line hairpin, a foldbackhairpin, a pendant hairpin, a chimera hairpin, a simplex free duplex, acircularized free duplex, a free hairpin duplex, and an anchored duplex.

Embodiment 3. The method of any one of embodiments 1 or 2, wherein thepulsed voltage is sufficient to release the duplex feature engaged withthe nanopore, while leaving the next duplex feature of the targetmolecule to engage with the nanopore unaffected.

Embodiment 4. The method of any one of embodiments 1-3, wherein thepulsed voltage has a duration of less than 100 microseconds, less than50 microseconds, less than 10 microseconds, less than 5 microseconds, orless than 1 microseconds.

Embodiment 5. The method of any one of embodiments 1-4, wherein thepulsed voltage has a voltage of greater than 0.2 volts, greater than 0.5volts, greater than 1 volt, or greater than 10 volts.

Embodiment 6. The method of any one of embodiments 1-4, wherein thepulsed voltage is 1 volt.

Embodiment 7. The method of any one of embodiments 1-6, wherein theperiodicity of the pulsed voltage ranges from 50 Hz to 10 kiloHz.

Embodiment 8. The method of any one embodiments 1-6, wherein theperiodicity of the pulsed voltage is 500 Hz.

Embodiment 9. The method of any one of embodiments 1-8, wherein thetarget molecule is sensed by the nanopore during the time period betweenpulses of the pulsed voltage.

Embodiment 10. The method of embodiment 3, wherein the duplex feature ofthe target molecule is released upon each pulse of the pulsed voltage.

Embodiment 11. The method of embodiment 3, wherein the duplex feature ofthe target molecule is released upon multiple pulses of the pulsedvoltage.

Embodiment 12. A reporter construct comprising a low impedance polymerand a high impedance polymer.

Embodiment 13. The reporter construct of embodiment 12, wherein the lowimpedance polymer comprises triethylene glycol (X), hexaethylene glycol(D), ethane (L), hexane (P), dodecane (Z), or abasic (Q), orcombinations thereof.

Embodiment 14. The reporter construct of embodiment 12 or 13, whereinthe high impedance polymer comprises deoxyadenosine (A), deoxythymine(T), deoxycytosine (C), or deoxyguanodine (G), or combinations thereof.

Embodiment 15. A reporter construct comprising two or morephosphoramidites selected from the group consisting of triethyleneglycol (X), hexaethylene glycol (D), ethane (L), hexane (P), dodecane(Z), deoxyadenosine (A), deoxythymine (T), deoxycytosine (C),deoxyguanodine (G) and abasic (Q), and wherein the reporter constructcomprises at least one of X, D, L, P, Z or Q.

Embodiment 16. The reporter construct of embodiment 15, comprising an A0reporter as set forth in FIG. 4.

Embodiment 17. The reporter construct of embodiment 15, comprising an A2reporter as set forth in FIG. 4.

Embodiment 18. The reporter construct of embodiment 15, comprising an A4reporter as set forth in FIG. 4.

Embodiment 19. The reporter construct of embodiment 15, comprising an A6reporter as set forth in FIG. 4.

Embodiment 20. The method of embodiment 1, wherein the target moleculecomprises a reporter construct of any one of embodiments 12-19.

The various embodiments described above can be combined to providefurther embodiments. All of the U.S. patents, U.S. patent applicationpublications, U.S. patent applications, foreign patents, foreign patentapplications and non-patent publications referred to in thisspecification and/or listed in the Application Data Sheet areincorporated herein by reference, including U.S. Provisional ApplicationNo. 61/996,824, filed on May 14, 2014, in their entirety. Aspects of theembodiments can be modified, if necessary to employ concepts of thevarious patents, applications and publications to provide yet furtherembodiments.

These and other changes can be made to the embodiments in light of theabove-detailed description. In general, in the following claims, theterms used should not be construed to limit the claims to the specificembodiments disclosed in the specification and the claims, but should beconstrued to include all possible embodiments along with the full scopeof equivalents to which such claims are entitled. Accordingly, theclaims are not limited by the disclosure.

1. A method for controlling the translocation of a target molecule forsensing by a nanopore, comprising passing the target molecule throughthe nanopore subjected to a base line voltage and a pulsed voltage,wherein the target molecule comprises two or more duplex features whichprovide translocation control by hybridization.
 2. The method of claim1, wherein the two or more duplex features which provide translocationcontrol by hybridization are selected from the group consisting of anin-line hairpin, a foldback hairpin, a pendant hairpin, a chimerahairpin, a simplex free duplex, a circularized free duplex, a freehairpin duplex, and an anchored duplex.
 3. The method of claim 1,wherein the pulsed voltage is sufficient to release the duplex featureengaged with the nanopore, while leaving the next duplex feature of thetarget molecule to engage with the nanopore unaffected.
 4. The method ofclaim 1, wherein the pulsed voltage has a duration of less than 100microseconds.
 5. The method of claim 1, wherein the pulsed voltage has avoltage of greater than 0.2 volts.
 6. The method of claim 1, wherein thepulsed voltage is 1 volt.
 7. The method of claim 1, wherein theperiodicity of the pulsed voltage ranges from 50 Hz to 10 kiloHz.
 8. Themethod of claim 1, wherein the periodicity of the pulsed voltage is 500Hz.
 9. The method of claim 1, wherein the target molecule is sensed bythe nanopore during the time period between pulses of the pulsedvoltage.
 10. The method of claim 3, wherein a duplex feature of thetarget molecule is released upon each pulse of the pulsed voltage. 11.The method of claim 3, wherein the duplex feature of the target moleculeis released upon multiple pulses of the pulsed voltage.
 12. A reporterconstruct comprising a low impedance polymer and a high impedancepolymer.
 13. The reporter construct of claim 12, wherein the lowimpedance polymer comprises triethylene glycol (X), hexaethylene glycol(D), ethane (L), hexane (P), dodecane (Z), or abasic (Q), orcombinations thereof.
 14. The reporter construct of claim 12, whereinthe high impedance polymer comprises deoxyadenosine (A), deoxythymine(T), deoxycytosine (C), or deoxyguanodine (G), or combinations thereof.15. A reporter construct comprising two or more phosphoramiditesselected from the group consisting of triethylene glycol (X),hexaethylene glycol (D), ethane (L), hexane (P), dodecane (Z),deoxyadenosine (A), deoxythymine (T), deoxycytosine (C), deoxyguanodine(G) and abasic (Q), and wherein the reporter construct comprises atleast one of X, D, L, P, Z or Q.
 16. The reporter construct of claim 15,comprising an A0 reporter as set forth in FIG.
 4. 17. The reporterconstruct of claim 15, comprising an A2 reporter as set forth in FIG. 4.18. The reporter construct of claim 15, comprising an A4 reporter as setforth in FIG.
 4. 19. The reporter construct of claim 15, comprising anA6 reporter as set forth in FIG.
 4. 20. (canceled)